Association of Monocyte Count With Lung Function and Exercise Capacity Among Hospitalized COVID‐19 Survivors: A 2‐Year Cohort Study

ABSTRACT Background Abnormal changes of monocytes have been observed in acute COVID‐19, whereas associations of monocyte count with long COVID were not sufficiently elucidated. Methods A cohort study was conducted among COVID‐19 survivors discharged from hospital. The primary outcomes were core symptoms of long COVID, distance walked in 6 min, and lung function, and the secondary outcomes were health‐related quality of life and healthcare use after discharge. Latent variable mixture modeling was used to classify individuals into groups with similar trajectory of monocyte count from discharge to 2‐year after symptom onset. Multivariable adjusted generalized linear regression models and logistic regression models were used to estimate the associations of monocyte count trajectories and monocyte count at discharge with outcomes. Results In total, 1389 study participants were included in this study. Two monocyte count trajectories including high to normal high and normal trajectory were identified. After multivariable adjustment, participants in high to normal high trajectory group had an odds ratio (OR) of 2.52 (95% CI, 1.44–4.42) for smell disorder, 2.27 (1.27–4.04) for 6‐min walking distance less than lower limit of normal range, 2.45 (1.08–5.57) for total lung capacity (TLC) < 80% of predicted, 3.37 (1.16–9.76) for personal care problem, and 1.70 (1.12–2.58) for rehospitalization after discharge at 2‐year follow‐up compared with those in normal trajectory group. Monocyte count at discharge showed similar results, which was associated with smell disorder, TLC < 80% of predicted, diffusion impairment, and rehospitalization. Conclusions Monocyte count may serve as an easily accessible marker for long‐term management of people recovering from COVID‐19.


| Introduction
COVID-19 is now recognized as a heterogeneous disease with a broad spectrum of symptom manifestations and varied disease severity, which is largely determined by variability of the host immune response following SARS-CoV-2 infection [1,2].Mononuclear phagocyte system (MPS), consisting mainly of monocytes and macrophages, has been reported to cause dysregulated immune response and the hyperinflammatory syndrome during acute COVID-19 infection [3,4].Previous studies about monocyte count with disease severity of COVID-19 during acute phase were inconsistent.Some studies with limited sample size found no significant differences in circulating monocyte counts among groups with different disease severity [5,6], whereas other studies found a decrease of monocyte count in the critical patients compared to mild and severe patients [7].Furthermore, an increase in classical monocyte and a decrease in nonclassical monocyte were found in peripheral blood of COVID-19 patients, which was considered to be key determinant of severe COVID-19 [5,7,8].The proportion of inflammatory monocyte-derived alveolar macrophage (MoAMs) was increased in bronchoalveolar fluid of severe COVID-19 patients compared with mild COVID-19 or healthy controls [9].Hence, abnormal changes of monocytes and MoAMs counts have been observed in acute COVID-19, but it is unclear whether they can return to normal during the recovery period in patients of varying severity and how long it will take.
In addition to host factors that determine disease severity at acute phase, circulating monocytes may also be related to host factors that can determine long-term consequences of COVID-19 [10,11], which was defined as "long COVID" or "post COVID condition" [12].The recovery process of multiple diseases including COVID-19 depends on MPS.Higher circulating monocyte counts were previously reported to be associated with shortened survival and disease progression in patients with fibrotic diseases, including idiopathic pulmonary fibrosis (IPF), systemic sclerosis, myelofibrosis, and hypertrophic cardiomyopathy [13,14].However, few follow-up studies conducted among COVID-19 survivors have focused on the longitudinal evolution of monocyte counts during convalescence and their association with long-term outcomes, including respiratory outcomes such as reduced lung function, aerobic capacity and endurance, quality of life, and healthcare use after discharge.
The aims of this study were to assess the relationships of monocyte count trajectories from discharge to 2-year follow-up with primary outcomes including core symptoms of long COVID, distance walked in 6 min, and lung function and the secondary outcomes including health-related quality of life (HRQoL) and healthcare use after discharge at 2 years after symptom onset among hospitalized COVID-19 survivors.Furthermore, the associations of monocyte count at discharge and highest monocyte count at acute phase with above outcomes were also explored.

| Study Design and Participants
Participants for the current study were from a cohort study of COVID-19 survivors discharged from hospital between January 7 and May 29, 2020.Inclusion and exclusion criteria of the cohort study have been described previously [15].Briefly, all patients with laboratory confirmed COVID-19 discharged from hospital between January 7 and May 29, 2020, were eligible for participation.Patients were excluded if they died after discharge and before first follow-up; were living in a nursing or welfare home; had psychotic disorder, dementia, or osteoarthropathy; or were immobile.Data at acute phase were retrieved from electronic medical records (Supporting Information S1: Methods Supplement).Three follow-up surveys were conducted at 6 months, 1 year, and 2 years after symptom onset.
Among 2469 participants with COVID-19, 2206 were eligible to be enrolled in the 2-year follow-up study, which was conducted at 2-year after symptom onset (Figure 1).Of these eligible participants, 1666 (75.5%) completed 2-year follow-up survey.After excluding participants without monocyte count value at acute phase (n = 33) or without monocyte count value after discharge (n = 244), the current analysis was restricted to 1389 participants.The study was approved by the Research Ethics Commission of hospital which enrolled the participants (KY-2020-78.01,KY-2020-78.03, and KY-2020-78.05).Written informed consent was obtained from COVID-19 survivors who attended the follow-up visit.

| Follow-Up Assessment
Eligible study participants were invited to attend face-to-face follow-up visits at the outpatient clinic of hospital at 6 months, 1 year, and 2 years after symptom onset.The 6-month, 1-year, and 2-year follow-up visits were conducted from June 16 to September 3, 2020, from December 16, 2020, to February 7, 2021, and from November 16, 2021, to January 10, 2022, respectively.A telephone survey was available for COVID-19 survivors at the 2-year follow-up visit as an alternative to the face-to-face interview, which was conducted by trained clinicians using the same questionnaires.The detailed 6-month 1-year, and 2-year follow-up procedures have been described previously [15][16][17].Briefly, at each visit, they underwent a detailed interview, a physical examination, and a 6-min walking distance (6MWD) test, and completed a series of questionnaires, including a self-reported symptom questionnaire, the modified British Medical Research Council (mMRC) dyspnea scale, the EQ-5D-5L questionnaire to assess HRQoL, the EuroQol Visual Analogue Scale (EQ-VAS; scores range from 0 to 100, with a higher score indicating a better health status), and so forth.Additionally, at the 1-year and 2-year visits, healthcare use after discharge was also collected by a questionnaire.Venous blood samples were drawn for the measurement of monocyte count and other laboratory indicators.A stratified disproportional random sampling procedure according to severity scale was used to select patients to receive pulmonary function tests (PFTs) at 6-month follow-up visit (Supporting Information S1: Methods Supplement).

| Outcomes
The primary outcomes were core symptoms more specifically related to long COVID (fatigue or muscle weakness, smell disorder, taste disorder, and dyspnea defined as mMRC ≥ 1) [18,19], distance walked in 6 min, and lung function (forced expiratory volume in 1 s [FEV 1 ] < 80% of predicted, forced vital capacity [FVC] < 80% of predicted, total lung capacity (TLC) < 80% of predicted, and diffusion capacity for carbon monoxide [DLCO] < 80% of predicted).The definition of fatigue or muscle weakness, smell disorder, and taste disorder was self-reported newly occurring or worse symptoms post COVID-19.The secondary outcomes were HRQoL (pain or discomfort, anxiety or depression, mobility, usual activity, and personal care) and healthcare use after discharge (outpatients' clinic visit, rehospitalization, and emergency department visit).

| Statistical Analysis
To identify different groups of study participants that share similar underlying trajectory of monocyte count from discharge to 2-year after symptom onset, latent variable mixture modeling was used to classify individuals into groups with similar patterns.The last monocyte count measured during hospitalization was defined as monocyte count at discharge.The median duration from measurement of last monocyte count during hospitalization to discharge was 4 (IQR 3-7) days.We fitted trajectory models and started with a 1-trajectory model in cubic form.The number of trajectories was determined by taking both the Bayesian information criterion for each set of trajectories and clinical implication into consideration.Finally, two trajectories with one in cubic form and the other in quadratic form were identified.
Demographic and clinical characteristics, clinical outcomes, and healthcare use after discharge of study participants were presented as median (IQR) for continuous variables and expressed as absolute values along with percentages for categorical variables.The comparisons of demographic and clinical characteristics, clinical outcomes, and healthcare use after discharge across different monocyte count trajectories were performed with χ 2 test, Fisher's exact, or Kruskal-Wallis test where appropriate.
Study participants were also categorized into three groups according to monocyte count at discharge (<0.40 × 10 9 /L, 0.40-<0.60× 10 9 /L, and ≥0.60 × 10 9 /L) and highest monocyte count at acute phase (<0.40 × 10 9 /L, 0.40-<0.60× 10 9 /L, and ≥0.60 × 10 9 /L), respectively.The comparisons of demographic and clinical characteristics, clinical outcomes, and healthcare use after discharge across these groups were performed with χ 2 test, Fisher's exact, or Kruskal-Wallis test where appropriate.The associations of monocyte count trajectories from discharge to 2-year follow-up with distance walked in 6 min and percentage of predicted distance walked in 6 min were assessed with multivariable adjusted generalized linear regression model.Multivariable adjusted logistic regression models were used to explore association of monocyte count trajectories with other outcomes, including core symptoms (fatigue or muscle weakness, smell disorder, taste disorder, and dyspnea), distance walked in 6 min less than lower limit of normal range (LLN), lung function (FEV 1 < 80% of predicted, FVC < 80% of predicted, TLC < 80% of predicted, and DLCO<80% of predicted), HRQoL (pain or discomfort, anxiety or depression, mobility, usual activity, and personal care), and healthcare use after discharge (outpatients clinic visit, rehospitalization, and emergency department visit).Age, sex, cigarette smoking (never-smoker, current smoker, and former smoker), education (college or higher vs. high school or lower), body mass index (BMI), comorbidity (hypertension, diabetes, cardiovascular diseases, cerebrovascular diseases, malignant tumor, chronic obstructive pulmonary disease, and chronic kidney disease), disease severity (scale 3, scale 4, and scale 5-6), and corticosteroids use during hospitalization were adjusted in these models.
The associations of monocyte count at discharge and highest monocyte count at acute phase with continuous and categorical outcomes were assessed with generalized linear regression models and logistic regression models, respectively.The covariables adjusted were the same as that adjusted in the modes for exploring association between monocyte count trajectories from discharge to 2-year follow-up and outcomes.
All significance tests were two-sided, and a p value less than 0.05 was considered statistically significant.All statistical analyses were done with SAS, version 9.4 (SAS Institute Inc, Cary, NC).

| Monocyte Count Trajectories From Discharge to 2-Year Follow-Up
As shown in Figure 2, two monocyte count trajectories from discharge to 2-year after symptom onset were identified.The sensitivity analysis after excluding participants with last monocyte count during hospitalization measured more than 7 days before discharge showed similar monocyte count trajectories from discharge to 2-year after symptom onset (Figure S1; Supporting Information S1: Results Supplement).

| Baseline Characteristics of Study Participants
The demographic and clinical characteristics of 1389 study participants included in this study are presented in Table 1.The median age of study participants was 57.0 (IQR 48.0-65.0)years, and 53% of them was male.Compared with study participants with normal monocyte count trajectory, those with high to normal high trajectory were more likely to be male and smokers, have hypertension, and receive corticosteroids and lopinavirritonavir during hospitalization.The study participants were not vaccinated at 6-month and 1-year follow-up, and 81% of them was vaccinated at 2-year follow-up.The proportion of vaccination was slightly higher among participants in normal trajectory group.Furthermore, BMI was higher, and length of intensive care unit (ICU) stay was longer among participants with high to normal high monocyte trajectory compared to those with normal monocyte trajectory.
The comparison of characteristics among participants classified by monocyte count at discharge and by highest monocyte count at acute phase both showed similar patter as the

| Clinical Outcomes and Healthcare Use of Study Participants
Table 2 shows the clinical outcomes and healthcare use of hospitalized COVID-19 survivors according to monocyte count trajectory from discharge to 2-year follow-up.The proportions of study participants with smell disorder, 6-min walking distance less than LLN, rehospitalization after discharge, and decreased TLC (<80% of predicted value) were higher among participants with high to normal high monocyte count trajectory compared to those with normal trajectory.Although the proportion of DLCO <80% of predicted was numerically higher among participants in the high to normal high trajectory group than those in the normal trajectory group (49% vs 43%), but the difference was not statistically significant (p = 0.51).Compared with participants with normal monocyte trajectory, EQ-VAS score was lower, whereas FEV 1 was higher among those with high to normal high monocyte trajectory.
The clinical outcomes and healthcare use of hospitalized COVID-19 survivors according to monocyte count at discharge and highest monocyte count during hospitalization were shown in Tables S3 and S4, respectively (Supporting Information S1: Results Supplement).

| Association of Monocyte Count Trajectories With Clinical Outcomes and Healthcare Use
The associations of monocyte count trajectories from discharge to 2-year after symptom onset with core symptoms, distance walked in 6 min, and lung function at 2-year follow-up are shown in Figure 3. Compared with participants in normal trajectory group, the multivariable adjusted odds ratios (ORs) (95% CIs) were 2.52 (  S5).
The sensitivity analyses with exclusion of participants with last monocyte count during hospitalization measured more than 7 days before discharge did not change the statistically significant association of monocyte count trajectory with smell disorder, 6-min walking distance less than LLN, and personal care problem (Figures S2 and S3).The associations of monocyte count trajectory with mobility problem and usual activity problem became statistically significant.
The sensitivity analyses did not substantially change the association between monocyte count at discharge and clinical outcomes and healthcare use, except that the association with rehospitalization after discharge was not statistically significant (Figures S2 and S3).

| Discussion
In the follow-up study of hospitalized COVID-19 survivors, we found survivors with high to normal high monocyte count trajectory from discharge to 2-year after symptom onset had a higher 2-year odds of smell disorder, poor exercise capacity, decreased TLC, poor HRQoL mainly in the domain of personal care, and all-cause rehospitalization after discharge compared with those with normal trajectory.Hospitalized COVID-19 survivors with monocyte count of ≥0.60 × 10 9 /L at discharge had higher 2-year  odds of smell disorder, decreased TLC, diffusion impairment, and all-cause rehospitalization after discharge versus those with monocyte count of <0.40 × 10 9 /L.To better elucidate the association of monocyte count with long-term outcomes among COVID-19 survivors, we further analyzed and summarized associations of monocyte count at all time points available in our study with outcomes at 2-year follow-up (Figure 5).The dynamic associations provided evidence for clinicians to make corresponding diagnosis and treatment decisions according to different monocyte level and prognosis status at different periods.
To our knowledge, this is the first study to describe the trajectory of monocyte count up to 2 years after SARS-CoV-2 infection.The levels of lymphocyte, neutrophil, and eosinophils at different follow-up time points were slightly higher among high to normal high trajectory group than that among normal trajectory group (Table S6).The persistent higher level of these peripheral blood markers may indicate a chronic inflammation status.Consistent with our findings, previous studies also found that abnormal peripheral blood monocyte count during acute phase of COVID-19 had returned to normal level during the convalescence [22,23], but when they will return to a baseline status before SARS-CoV-2 infection was unclear.Elevated monocytes after acute SARS-CoV-2 infection were recruited to the airways to differentiate into monocyte-derived alveolar macrophage, which has been reported to be associated with pulmonary fibrosis by stimulating and forming reciprocal circuits with fibroblasts [24][25][26].In patients with IPF, elevated monocyte count was associated with increased risks of IPF progression, accelerated decline in lung function, hospitalization, and mortality, which indicated monocyte count may serve as a simple and inexpensive prognostic biomarker [13,14,20,27].However, the role of monocyte count played in the long-term recovery process of COVID-19 patients was not clearly delineated.Better understanding the association of post-acute monocyte count recovery trajectories rather than just focusing monocyte count at acute phase would be more enlightening for long-term management of people recovering from COVID-19.
Long COVID has become a global concern, and its underlying pathogenesis was still unclear.One possible reason may be due to the chronic inflammation caused by persistent SARS-CoV-2 virus or viral antigens and RNA in multiple tissues [2,21].Pathogen-associated molecular patterns caused by persistent SARS-CoV-2 reservoir or remnants could engage various host pattern recognition receptors to trigger innate immune activation [21].Recently, SARS-CoV-2 antigen and RNA were found in tissue macrophages of recovered patients around 1 year after symptom onset [28,29].This may have some influence on the stable state of peripheral circulation of monocytes.The current finding of association of monocyte count trajectory from discharge to 2year follow-up, monocyte count at discharge, and highest monocyte count at acute phase and worse outcomes (results for highest monocyte count at acute phase shown in Figures S4 and S5 Our study has several limitations.First, we mainly focused on monocyte counts without evaluating monocyte function; thus, further studies are needed to explore association of monocyte subset and monocyte activation markers with long-term outcomes.Second, this is a single-center study with enrollment of COVID-19 survivors who discharged at the early stage of pandemic, which may limit the generalizability of study findings.Finally, information bias resulted from recalling information such as smoking and self-reported comorbidity cannot be excluded, even though misclassification of these variables was more likely to be non-differential in our cohort study.
The cohort study with 2-year follow-up duration after symptom onset among people recovering from COVID-19 provided evidence for the association of both monocyte count trajectory from discharge to follow-up and elevated monocyte counts at discharge with increased risk of worse physical or functional outcomes among COVID-19 survivors.The findings provided clues for monocyte count as an easily accessible marker for long-term management of people recovering from COVID-19.Future studies with longer follow-up duration and larger sample size are needed to more comprehensively understand the role of monocyte served for pathophysiology of long COVID, with focusing on not only monocyte count but also its function.

FIGURE 1 |
FIGURE 1 | Flow chart of study participants.

FIGURE 2 |
FIGURE 2 | Trajectories of monocyte count from discharge to 2 years after symptom onset among hospitalized COVID-19 survivors.
) in COVID-19 survivors provided us a new insight into the use of monocyte count for identification of COVID-19 survivors at risk of poor long-term prognosis.Evaluation of monocyte count and its recovery trajectory, as an easily accessible resource, provides the first indicator of long-term outcome in the absence of functional information among people recovering from COVID-19.

FIGURE 3 |FIGURE 4 |
FIGURE 3 | Association of monocyte count trajectories and monocyte count at discharge with core symptoms (a), distance walked in 6 min (b) and lung function (c) at 2-year follow-up.

TABLE 1 |
Baseline characteristics of hospitalized COVID-19 survivors according to monocyte count trajectories.

TABLE 2 |
[20,21]l outcomes and healthcare use of hospitalized COVID-19 survivors at 2-year follow-up visits according to monocyte count trajectories.EuroQol Visual Analogue Scale; FEV 1 = forced expiratory volume in 1 s; FVC = forced vital capacity; LLN = lower limit of normal range; mMRC = modified British Medical Research Council; TLC = total lung capacity. a Core symptoms were identified as symptoms more specifically related to long COVID according to findings of Mizrahi and Ballering[20,21].bPredictedvalues were calculated according to the method of Enright and Sherrill.cThelower limit of the normal range was calculated by subtracting 153 m from the predicted value for men or by subtracting 139 m for women.